DNA Methylation

• Epigenetics: the passing on of modified inheritable traits to subsequent generations without changing the DNA sequence per se... Epigenetic trait modification can be triggered by environmental factor(s).

• Pembrey (a geneticist at University College London in the U.K.) and his colleagues (who studied the unusually detailed historical medical records of the isolated northern Swedish city of Overkalix) found that grandsons of men who experienced famine during mid-childhood went through puberty earlier and had longer lifespans, while the grandsons of men who were well fed in early childhood had an increased likelihood of diabetes.

• For females, the effect was similar but it was tied to the grandmother, rather than the grandfather.

• More contemporaneously, Pembrey has found that fathers who had started smoking before age 11 had sons who were significantly fatter than average. There was no similar effect on daughters Figure 1. Epigenetic inheritance via the female and male germ line. If a gestating mother (F0, blue) is exposed to environmental stressors, her fetus (green) and its already developing germ line (red) are also directly exposed. As a result, phenotypes observed up to the F2 generation (red) may result from the grandmother’s experience. The F3 generation (yellow) is the frst generation that has not been exposed to these environmental stressors, thus phenotypes observed in this generation could represent transgenerational epigenetic inheritance. If inheritance via the male or the maternal germ line before gestation is investigated, environmental stressors may affect the F0 generation (blue) and the developing germ line (red). As a result, the frst generation that could represent transgenerational epigenetic inheritance is F2 (yellow).

• Angelman syndrome results from a loss of gene activity in a specifc part of chromosome 15 (the 15q11-q13 region).

– This region contains a gene called UBE3A that, when mutated or absent, likely causes the characteristic features of this condition. People normally have two copies of the UBE3A gene, one from each parent. – Both copies of this gene are active in many of the body's tissues. – In the brain, however, only the the maternal copy is active. If the maternal copy is lost because of a chromosomal change or a gene mutation, a person will have no working copies of the UBE3A gene in the brain.

• Prader-Willi syndrome is caused by the loss of active genes in a specifc part of chromosome 15, the 15q11-q13 region. People normally have two copies of this chromosome in each cell, one copy from each parent. – Prader-Willi syndrome occurs when the paternal copy is partly or entirely missing

6 Epigenetics

• Epigenetics: the passing on of modified gene functions to subsequent generations without changing the underlying DNA sequence. ... Epigenetic modifications can be triggered by environmental factors.

• For females, the effect was similar but it was tied to the grandmother, rather than the grandfather.

• More contemporaneously, Pembrey has found that fathers who had started smoking before age 11 had sons who were significantly more obese than average. There was no similar effect on daughters Epigenetic Effects Can Be Inherited

• Epigenetic effects can result from modification of a nucleic acid after it has been synthesized or by the perpetuation of protein structures.

• Epigenetic effects may be inherited through generations (transgenerational epigenetics). • The two pictures show the eyes of two genetically identical flies. The difference in eye colour is determined by epigenetic factors. (Credit: Renato Paro/ETH Zürich)

9 Epigenetic affects of maternal diet...

ScienceDaily (Mar. 9, 2011) — Poor diet during pregnancy increases offspring's vulnerability to the effects of aging, new research has shown for the first time.

To test their theory, the researchers used a well-established rat model where, by altering the protein content of the mother's diet during pregnancy, the offspring develop type 2 diabetes in old age.

First, they studied the RNA from insulin secreting cells in the pancreas from offspring of normally fed as well as malnourished mothers in young adult life and in old age. When they compared the two, they found that there was a significant decrease in the expression of the Hnf4a gene in the offspring prone to type 2 diabetes. The expression of Hnf4a also decreased with age in both groups.

Second, they studied the DNA and found that the decrease of Hnf4a was NOT caused by any genetic changes, but rather by some epigenetic changes...... Caring Mothers Reduce Response to Stress for Life

How a rat responds to stress depends on whether its mother cared for it properly as a pup, which marks its genes for life. Dr. Mae-Wan Ho reports

Maternal effects in the spotlight

Maternal effects on the development of offspring are well known. But they are thought to be due to nutritional and physiological factors affecting the foetus in the womb; and within the past few years, geneticists have discovered that diet and stress can profoundly change the pattern of gene expression in the offspring, affecting their health prospects as adults (see Diet trumping genes, SiS 20).

A team of researchers from the Douglas Hospital Research Centre and McGill University in Montreal Canada, and the Molecular Medicine Centre, in Edinburgh University Western General Hospital in the UK, now report a remarkable experiment in which the behaviour of the mother nursing her pups not only affects the pups’ response to stress as adults, but are correlate with changes in gene expression states in brain cells that persist into adult life. Such changes are referred to as ‘epigenetic’ as they do not involve alterations in the base sequence of DNA in the genome, only their off and on states; but they can persist in the brain cells and are passed on to all the daughter cells. Caring mothers reduce stress response of pups

In the nest, the mother rat licks and grooms her pups and, while nursing, arches her back to groom and lick her pups. Some mothers (high performers) tend to do these acts more frequently than others (low performers). As adults, the offspring of high performers are less fearful and show more modest responses to stress in the hypothalamus-pituitary-adrenal (HPA) neuro-endocrine pathway. ISIS Report113 07/09/04 Four Recognized Mechanisms of Cellular Epigenetc Inheritance Systems

• Self sustaining feedback loops • 3 D templatng -Structural Inheritance • Chromatn Marking • RNA-mediated EIS

12 Four Recognized Mechanisms of Cellular Epigenetc Inheritance Systems

• Self sustaining feedback loops • 3 D templatng -Structural Inheritance • Chromatn Marking • RNA-mediated EIS

13 Four Recognized Mechanisms of “Cellular Epigenetc Inheritance Systems” • Self sustaining feedback loops -protein products (or modifcatons thereof) or mRNA concentratons… – Candida albicans, where an epigenetc switch underlies the transiton between white and opaque cells—two states that are heritable for many generatons due to the presence of a protein (Wor1) that gets diluted over generatons… leading to a change of cell type back to “white”.

• 3 D templatng -Structural Inheritance • Chromatn Marking • RNA-mediated EIS

14 15 Four Recognized Mechanisms of Cellular Epigenetc Inheritance Systems

• Self sustaining feedback loops • 3 D templatng -Structural Inheritance – Inheritance of alternatve three-dimensional (3-D) structures through spatal templatng: Prions • Chromatn Marking • RNA-mediated EIS

16 • prion – A proteinaceous infectious agent that behaves as an inheritable trait, although it contains no nucleic acid. – Examples are:

– Bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, and Scrapie in sheep a both fatal, neurodegenerative diseases that cause a spongy degeneration in parts of the brain and spinal cord -caused by an abherant form of a protein PrPSc.

– Sup35 in Yeast

– PrPSc, the agent of scrapie in sheep and bovine spongiform encephalopathy in cattle, and Psi, which confers an inherited state in yeast. Yeast Prions Show Unusual Inheritance

• amyloid fibers – Insoluble fibrous protein polymers with a cross β-sheet structure, generated by prions or other dysfunctional protein aggregations (such as those fibrous protein polymers present in Alzheimer’s patients).

• Conversion between the two forms of a given protein -can be influenced by action of “chaperones”.

• The wild-type form has the recessive genetic state psi– and the mutant form has the dominant genetic state PSI+. Yeast Prions Show Unusual Inheritance

• The Sup35 protein in its wild-type soluble form is a termination factor for translation. • Sup35 can also exist in an alternative form of oligomeric aggregates, in which it is not active in protein synthesis.

The [PSI+] state is due to protein aggregation as a function of the history of the strain Yeast Prions Show Unusual Inheritance

• The presence of the oligomeric form causes newly synthesized protein to acquire the inactive structure.

The [PSI+] state is dominant or recessive ? Prions Cause Diseases in Mammals

• Scrapie (in sheep) is a neurodegenerative disorder that is manifested by the inability of the sheep to remain standing upright.

• The protein responsible for scrapie exists in two forms and is normally expressed in the brain:

• the Wild-Type, non-infectious form PrPC is susceptible to proteases, while the disease- causing form PrPSc is resistant to proteases

• Difference in these two forms appears to lie with a change in conformation rather than “conventional” alterations (i.e. methylation, acetylation, sequence mutation)

• The PrPSc protein can perpetuate itself by causing the newly synthesized PrP protein to take up the PrPSc form instead of the PrPC form

• Different (multiple, variant) strains of prions may interact, leading to the formation of many different transmissible (cell-heritable and infectious) phenotypes

• Indeed, multiple strains of PrPSc may have different conformations of the protein -giving rise to variable degrees of severity of the phenotype. Prions Cause Diseases in Mammals

• The neurological disease, scrapie is also in mice and can be transmitted to mice by injecting the purified PrPSc protein into mice.

• However, the recipient mouse must already have a copy of the PrP gene, coding for the mouse protein, present.

PrP interactivity is not always restricted to the “same” species Prions Cause Diseases in Mammals

• The neurological disease, scapie can be transmitted to mice by injecting the purified PrPSc protein into mice.

• The recipient mouse must

PrP interactivity is not always restricted to species For Recognized Mechanisms of Cellular Epigenetc Inheritance Systems

• Self sustaining feedback loops • 3 D templatng -Structural Inheritance • Chromatn Marking – Physical changes in DNA, histones and non-histonal proteins interactng with chromatn caused by chemical modifcaton – Numerous examples. • RNA-mediated EIS

Methylation requires not only de novo activity, but also maintenance following replication

The fidelity of transmission in cell lineages ranges from about 1% variation per cell generation up to the very high fidelity of 10-6 variations per cell generation • nature nature reviews genetics review articles article • Published: 30 May 2017 TET-mediated active DNA demethylation: mechanism, function and beyond • Xiaoji Wu & Yi Zhang Nature Reviews Geneticsvolume 18, pages 517–534(2017)

Abstract In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodifed cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation. Additionally, many regulatory mechanisms of this process have been identifed. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions.

27 28 DNA methylation is probably the best- understood system of Epigenetic inheritance as it is a genetic modifcation found in Eubacteria, Archeabacteria, and Eukaryota. It is involved in many important functions In eukaryotes, methylation usually occurs on the cytosines in CpG doublets and also in CpNpG triplets in plants. Since CpG and CpNpG are palindromes… they provide perfect template for “mirroring” regulatory markers one at of the DNA strands.

The typical density of CpG doublets in mammalian DNA is ~1/100 bp, as seen for alpha -globin gene. Epigenetic Efects

The ability of different states to be inherited without any change in sequence of DNA.

Typically, these different states result in different phenotypes.

These state can result from action of Protein aggregates that control the conformation of new subunits as they are synthesized

Modification of a nucleic acid AFTER it has been synthesized by the perpetuation of protein structures that may assemble on the DNA Methylation requires maintenance methylation following replication… but also de novo activity.

The fidelity of methyl transmission in cell lineages ranges from about 1% variation per cell generation up to the very high fidelity of 10-6 variations per cell generation

Epigenetic Efects

The ability of different states to be inherited without any change in sequence of DNA.

Typically, these different states result in different phenotypes.

These state can result from action of Protein aggregates that control the conformation of new subunits as they are synthesized

Modification of a nucleic acid AFTER it has been synthesized by the perpetuation of protein structures that may assemble on the DNA

Histone tails have many sites of modification

Adapted from The Scientist 17 (2003): p. 27. Most modified sites in histones have a single, specific type of modification, but some sites can have more than one type of modification 39 Acetylation during replication occurs on specific sites on histones before they are incorporated into nucleosomes Acetylation associated with gene activation occurs by directly modifying specific sites on histones that are already incorporated into nucleosomes

45 Angiotensin-converting enzyme (ACE) inhibitors help relax your veins and arteries to lower your blood pressure. ACE inhibitors prevent an enzyme in your body from producing angiotensin II, a substance that narrows your blood vessels. This narrowing can cause high blood pressure and force your heart to work harder.

ACE2 inactivates angiotensin II and is a negative regulator of the system. 46

Nature volume 436, pages 112–116 (2005) Angiotensin-converting enzyme 2 (ACE2), the proposed receptor of SARS- CoV-2, is expressed in the respiratory airways at low levels (blue) compared to the intestine, kidney, heart, and pancreas. Figure 1. The expression of the ACE2 and interferon gene depends on the methylation rate of the CpG islands in the DNA promotor sequence.

Susceptible individuals, mostly men, the elderly, and smokers, show a hypomethylation pattern of the ACE2 and interferon genes (lower part), whereas women, children, and non-smokers show DNA hypermethylation and lower expression of ACE2 and interferon proteins (upper part).

The higher presence of ACE2 on epithelial cells and interferon makes people more susceptible to SARS-CoV-2 infection and increases disease severity, whereas a low presence of ACE2 and interferon seems to offer disease protection

In this study, 700 lung transcriptome samples of patients with comorbidities and suffering from severe SARS-CoV-2 were analyzed, and it was found that ACE2 was highly expressed in these patients compared to control individuals (Pinto et al., 2020).

MINI REVIEW ARTICLE Front. Cell. Infect. Microbiol., 26 May 2020 | https://doi.org/10.3389/fcimb.2020.00290 Histone Acetylation Is Associated with Transcription Activation

• Histone acetyltransferases (HATs) vary in their target specificity.

• Deacetylation is associated with repression of gene activity.

• histone deacetylases (HDAC’s) – Enzymes that removes acetyl groups from histones; may be associated with repressors of transcription.

• Deacetylases are very often present in complexes with repressor activity. Modifying complexes have several components • Figure 2. Proposed mechanism by which DNA methylation leads to transcriptional repression. • (a) Transcriptionally active chromatin is predominantly unmethylated and has high levels of acetylated histone tails (short black squiggles). • (b) Methylation at CpG dinucleotides can be carried out by one of the three known human DNA methyltransferases (DNMT1, 3a and 3b), resulting in DNA with high levels of CpG methylation (purple circles), but still containing predominantly acetylated histone tails. DNA in this form would still be expected to be transcriptionally competent. • (c) Methylated DNA is targeted by methyl-binding domain (MBD) proteins such as MBD2 and MeCP2, which are found associated with large protein complexes such as the NuRD complex (MBD2) and the Sin3a complex (MeCP2). • Histone deacetylase (HDAC1 and 2) and chromatin- remodelling activities (Mi-2 and Sin3a) within these complexes result in alterations in chromatin structure, producing chromatin that is refractory to transcriptional activation (pink streaks represent deacetylated histone tails).

Proposed mechanism by which DNA methylation leads to transcriptional repression

G. Strathdee and R. Brown 51 • Figure 2. Proposed mechanism by which DNA methylation leads to transcriptional repression. • (a) Transcriptionally active chromatin is predominantly unmethylated and has high levels of acetylated histone tails (short black squiggles). • (b) Methylation at CpG dinucleotides can be carried out by one of the three known human DNA methyltransferases (DNMT1, 3a and 3b), resulting in DNA with high levels of CpG methylation (purple circles), but still containing predominantly acetylated histone tails. DNA in this form would still be expected to be transcriptionally competent. • (c) Methylated DNA is targeted by methyl-binding domain (MBD) proteins such as MBD2 and MeCP2, which are found associated with large protein complexes such as the NuRD complex (MBD2) and the Sin3a complex (MeCP2). • Histone deacetylase (HDAC1 and 2) and chromatin- remodelling activities (Mi-2 and Sin3a) within these complexes result in alterations in chromatin structure, producing chromatin that is refractory to transcriptional activation (pink streaks represent deacetylated histone tails).

Proposed mechanism by which DNA methylation leads to transcriptional repression

G. Strathdee and R. Brown 52 • Figure 2. Proposed mechanism by which DNA methylation leads to transcriptional repression. • (a) Transcriptionally active chromatin is predominantly unmethylated and has high levels of acetylated histone tails (short black squiggles). • (b) Methylation at CpG dinucleotides can be carried out by one of the three known human DNA methyltransferases (DNMT1, 3a and 3b), resulting in DNA with high levels of CpG methylation (purple circles), but still containing predominantly acetylated histone tails. DNA in this form would still be expected to be transcriptionally competent. • (c) Methylated DNA is targeted by methyl-binding domain (MBD) proteins such as MBD2 and MeCP2, which are found associated with large protein complexes such as the NuRD complex (MBD2) and the Sin3a complex (MeCP2). • Histone deacetylase (HDAC1 and 2) and chromatin- remodelling activities (Mi-2 and Sin3a) within these complexes result in alterations in chromatin structure, producing chromatin that is refractory to transcriptional activation (pink streaks represent deacetylated histone tails).

Proposed mechanism by which DNA methylation leads to transcriptional repression

G. Strathdee and R. Brown 53 • Figure 2. Proposed mechanism by which DNA methylation leads to transcriptional repression. • (a) Transcriptionally active chromatin is predominantly unmethylated and has high levels of acetylated histone tails (short black squiggles). • (b) Methylation at CpG dinucleotides can be carried out by one of the three known human DNA methyltransferases (DNMT1, 3a and 3b), resulting in DNA with high levels of CpG methylation (purple circles), but still containing predominantly acetylated histone tails. DNA in this form would still be expected to be transcriptionally competent. • (c) Methylated DNA is targeted by methyl-binding domain (MBD) proteins such as MBD2 and MeCP2, which are found associated with large protein complexes such as the NuRD complex (MBD2) and the Sin3a complex (MeCP2). • Histone deacetylase (HDAC1 and 2) and chromatin- remodelling activities (Mi-2 and Sin3a) within these complexes result in alterations in chromatin structure, producing chromatin that is refractory to transcriptional activation (pink streaks represent deacetylated histone tails).

Proposed mechanism by which DNA methylation leads to transcriptional repression

G. Strathdee and R. Brown 54 55 Trends Genet. 2011 Mar; 27(3):56 116–125. Trends Genet. 2011 Mar; 27(3):57 116–125. Trends Genet. 2011 Mar; 27(3):58 116–125. Trends Genet. 2011 Mar; 27(3):59 116–125. Trends Genet. 2011 Mar; 27(3):60 116–125.

Epigenetic differences arise in MZ twins.

Fraga M F et al. PNAS 2005;102:10604-10609

(A) Two representative examples of the determination of monozygosity using microsatellite markers.

(B) Quantification of X chromosome inactivation by PCR amplification of the androgen receptor locus after digestion with the DNA methylation- sensitive (HpaII) and -insensitive restriction enzymes (MSp I), respectively. Two examples of a different pattern of X inactivation between MZ twin pairs are shown.

(C)(C Upper) Quantification of global 5mC DNA content (Left), histone H4 acetylation (Center), and histone H3 acetylation (Right) by HPLC and high-performance capillary electrophoresis. (C Lower) Comparison of epigenetic values between the siblings of each 3- and 50-year-old twin pair. Results are expressed as mean ± SD. Trends Genet. 2011 Mar; 27(3):64 116–125. There is evidence that the rate of transmission of epigenetic marks lessens with each generation [57], suggesting that epigenetic profiles are likely to be more similar in families within generations as opposed to between generations This would be in contrast to transgenerational inheritance of DNA methylation patterns in plants, which can be relatively stable for up to eight generations [48,50],

Trends Genet. 2011 Mar; 27(3):65 116–125.

Chromatin Remodeling is a Necessary and Active Process

• chromatin remodeling – The energy-dependent displacement or reorganization of nucleosomes that occurs in conjunction with activation of genes for transcription.

Chromatin structure is stable • A remodeling complex does not itself have any specificity for any particular target site, and must be recruited by a component of the transcription apparatus.

• Remodeling complexes are recruited to promoters by sequence-specific activators.

• The factor may be released once the remodeling complex has bound.

Remodeling complexes bind via activators • All remodeling complexes contain a related ATPase catalytic subunit, and are grouped into subfamilies containing more closely related ATPase subunits.

• Remodeling complexes can alter, slide, or displace nucleosomes.

• Some remodeling complexes can exchange one histone for another in a nucleosome.

Remodeling changes nucleosome organization • There are numerous ATP- dependent chromatin remodeling complexes that use energy provided by hydrolysis of ATP.

Nucleosome displacement is an active process • a, In the off state, the DNA-bound repressor (REP) at the upstream repressor site (URS) recruits negative modifiers, such as histone deacetylase (HDAC), which remove acetyl (ac) groups from histones. • b, In the on state, DNA-bound activator (ACT) at the upstream activator site (UAS) recruits positive modifiers, such as histone acetylases (HAT), at the promoter, while DNA-bound RNA polymerase (POL) recruits histone methylases at the ORF. Early during elongation, the C- terminal domain (CTD) polymerase repeat is phosphorylated at serine 5 (S5ph), leading to recruitment of the COMPASS complex (Set1, part of the COMPASS complex, methylates H3K4) and DOT1 (which methylates H3K79). Later in elongation the CTD repeat is phosphorylated at 42 serine 2 (S2ph), leading to recruitment of Set2 (which methylates H3K36). 32